Pathogenic bacteria are responsible for millions of deaths every year with an estimated mortality of 70 million people by 2010 for Mycobacterium tuberculosis alone. Novel methods for identification of bacterial species in hosts, urban environments, water sources and food stuffs are required to advance diagnosis and preventative medicine. Detection of bacterial species in environmental samples is a complex task since large numbers of bacteria are present and are resistant to culturing. Therefore, the genetic content of the entire sample has to be analysed simultaneously and this constitutes a metagenomic sample. Commonly-used methods of bacterial identification focus on detection of specific genomic regions to determine species. Currently only one percent of a metagenomic sample can be used for identification employing phylogenetic markers. This method is highly inefficient. The search for more widespread markers within each genome is essential to improve detection methods. Also, modern sequencing technologies used in these environments have short read lengths which prove difficult to assemble e.g. repeats can lead to incorrect assembly. The use of overrepresented oligonucleotides provides a solution to both of these difficulties. Overrepresented oligonucleotides (8-14bp in length) are utilised to differentiate between species based on observed frequency of occurrence rather than presence or absence. They occur throughout the genome thereby increasing genomic coverage. Furthermore, overrepresented oligonucleotides can be easily identified in a raw metagenomic sample, bypassing the need for sequence assembly. Raw oligonucleotide data was filtered, analysed and imported into a structured database. A program, Oligosignatures, allowed for creation of species and phylogenetic lineage specific oligonucleotide markers dependent on the selection of species specified by the user. For the purposes of this study, the context of bacterial identification in an unknown environment was selected. A similarity trial was then executed to determine if strains of the same species can be separated from each other using overrepresented oligonucleotides. Outcomes of this test provided a guideline for the creation of species and lineage specific oligonucleotide markers. Each species and lineage was therefore described by a marker profile which consisted of representative oligonucleotide markers. These marker profiles were then tested against artificial and experimental data to determine their effectivity. Two approaches were used for testing, namely Oligonucleotide frequency analysis and Sequence read analysis. Oligonucleotide frequency analysis focused on the identification of species dependent on the global frequencies of marker oligonucleotides within each marker profile. Sequence read analysis attempted to assign metagenomic reads to a specific species dependent on the number of marker oligonucleotides present within the read. The final database contained 439 bacterial genomes from 22 different phylogenetic lineages. Interpretation of the results obtained after strain similarity testing showed that strains of the same species had highly similar markers and were not separable using this approach. All strains of a species that conformed to this premise were reduced to a single representative member. Similarly, species marker profiles demonstrated that closely related species remained difficult to separate. Twenty-one of the 22 lineages showed sufficient lineage specific markers for use in testing. This provides support for the abundance of overrepresented oligonucleotides and their potential for use as a detection method. In general, metagenomic testing of marker profiles showed that species specific determination was prone to interference, specifically, in closely related species. However, more distantly related species could be separated using both methods. Lineage discrimination generated more reliable results proving that lineage determination was possible in both artificial and experimental datasets. Oligonucleotide frequency analysis, the most sensitive approach, showed the best results for lineage determination but poorer results for species identification. Sequence read analysis provided a more effective method of determining confidence using different thresholds for read classification. In conclusion, the use of overrepresented oligonucleotides holds promise as a novel method for bacterial identification in a metagenomic context. Although several obstacles still prevent optimal utilization of these oligonucleotides, with further research the classification and identification of species and phylogenetic lineages from metagenomic samples can become a reality. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Biochemistry / unrestricted

Les microorganismes représentent la forme de vie la plus diverse et abondante sur Terre et jouent un rôle fondamental dans tous les processus biologiques. Cependant, du fait de la grande diversité des communautés microbiennes, la caractérisation fine des environnements complexes reste difficile par les approches moléculaires actuelles de PCR et de métagénomique. En effet, ces approches ne conduisent qu’à une caractérisation partielle des communautés et ne permettent pas systématiquement d’associer la structure des communautés aux fonctions métaboliques réalisées. L’approche de capture de gènes par hybridation appliquée à des échantillons métagénomiques complexes a démontré son intérêt pour révéler toute la diversité connue mais aussi inconnue des biomarqueurs fonctionnels ciblés, ainsi que pour enrichir leurs régions flanquantes sur quelques centaines de permettant en évidence des associations de gènes. Ainsi, les travaux de thèse ont visé à développer une nouvelle méthode de capture de gènes par hybridation capable d’enrichir de façon ciblée de larges régions génomiques à partir d’échantillons complexes, permettant ainsi de faire le lien entre structure et fonction des communautés microbiennes. Ces développements ont nécessité la détermination de sondes de capture, l’utilisation d’une méthode d’extraction d’ADN de haut poids moléculaire et la mise au point d’un protocole de capture permettant de piéger des fragments nucléiques de grande taille (jusqu’à 50 kb). La validation de la méthode de capture par hybridation sur un échantillon environnemental de sol a permis de révéler tout son potentiel. Appliquée au gène exprimant l’ARNr 16S, cette stratégie a permis de révéler une diversité microbienne non accessible par les approches moléculaires conventionnelles, avec une résolution d’identification jusqu'au niveau de l’espèce rendue possible grâce à la reconstruction de la séquence complète de ce marqueur phylogénétique. Appliquée à un gène fonctionnel, elle a conduit à la reconstruction de la séquence du biomarqueur et de ses régions flanquantes pouvant atteindre plusieurs dizaines de kb, permettant d’identifier les microorganismes possédant les capacités métaboliques d’intérêt. Ainsi, la capture par hybridation représente une approche alternative prometteuse pour le diagnostic environnemental en conduisant à une meilleure caractérisation des communautés microbiennes. / Microorganisms are the most diverse and abundant life forms on Earth and are key players in thefunctioning of all biological processes. Nevertheless, PCR and metagenomics strategies aiming to describemicrobial communities are hampered by their huge diversity. Indeed, these molecular methods only drive to apartial description of communities and do not systematically allow linking functions back to the identities of themicroorganisms. Hybridization capture applied to complex metagenomic samples has demonstrated its efficiency to reveal all known and unknown diversity of targeted biomarkers, and to enrich their flanking regions over a few hundred bp facilitating the discovery of gene associations.Thus, this work aimed at developing a new hybridization capture method capable of specifically enrichinglarge genomic regions from complex samples allowing to associate structure and functions of communities. Thedevelopment of this method required the design of capture probes, the use of a high molecular weight DNAextraction method, and the elaboration of a capture protocol dedicated to the enrichment of large genomicfragments (up to 50 kbp).The validation of the hybridization capture method on an environmental soil sample uncovered all itspotential. Applied to the 16S rRNA gene, this strategy revealed greater microbial diversity than conventionalmolecular methods and improved phylogenetic resolution up to the species level thanks to the reconstruction offull-length genes. Applied to a functional gene, the method enabled the reconstruction of large genomic regionscarrying the targeted biomarker and its flanking regions over several tens of kbp, leading to the identification ofmicroorganisms with specific metabolic functions. Hybridization capture thus appears as a promising alternativemethod for environmental diagnosis, through providing a better knowledge of microbial communities.